Abstract Necroptosis, in which the FADD-caspase-8-FLIP complex controls RIPK3-MLKL-mediated cell death, is an inflammatory cell death process that is engaged by TNF and related ligands, TLR signaling (involving TRIF) and interferons. Necroptosis has been implicated in development and adult homeostasis. Through genetic models and in vitro cell-based experiments, we have learned that RIPK1 plays contrasting roles in necroptosis, either promoting or inhibiting the process in different settings. Our central hypothesis, based on our published and extensive preliminary data, is that the interaction of FADD-caspase-8-FLIP with RIPK1-RIPK3-MLKL is fundamentally important in the regulation of inflammatory diseases. Based on this hypothesis, we will ask: 1. How do FADD-caspase-8-FLIP and RIPK1 function in the regulation of RIPK3? Here we will explore the roles of RIPK1 in positively and negatively regulating RIPK3 and necroptosis, both by recruiting FADD- caspase-8-FLIP to the necrosome complex and the inhibitory function of its kinase-inactive form. Cell-based in vitro studies will elucidate how RIPK1 performs these functions, and we will apply these to our paradoxical finding that the kinase-inactive mutant of RIPK1 may not rescue the embryonic lethality of FADD- or caspase- 8-deficient mice. 2. How do the survival functions of FADD-caspase-8-FLIP and RIPK1 function in inflammatory disease? Here we will employ novel genetic models (mice with a floxed allele of RIPK3, mice harboring a caspase-8 mutant with the survival but not the apoptotic function of caspase-8) to explore several inflammatory conditions in which involvement of apoptosis or necroptosis has been suggested. 3. How do FADD-caspase-8-FLIP and RIPK3-MLKL suppress ALPS? While autoimmune lymphoproliferative syndrome (ALPS) has been studied for decades in the context of CD95/CD95L defects, little is known about the pathogenesis of this disease, largely due to an inability of CD95-deficient bone marrow to transfer the syndrome. In contrast, we have found that ALPS in casp8-/-ripk3-/- animals is readily transferred to wt recipients. We will exploit this, and our novel genetic models, in the exploration of this syndrome. The proposed studies, throughout, are designed to provide a deeper understanding into the functions of RIPK1 interactions with FADD-caspase-8-FLIP, and RIPK3-MLKL, two interacting pathways to different forms of cell death. Through these ongoing investigations, we will extend our fundamental insights into how the regulation of the RIPK1-RIPK3-MLKL axis by the function of FADD-caspase-8-FLIP impacts on inflammatory diseases.